Ryouta Maeba

Oxidized low-density lipoprotein induces the production of superoxide by neutrophils

Exposure of guinea pig peritoneal neutrophils to ox‐LDL led to the production of superoxide, which was measured by the formation of superoxide‐dependent chemiluminescence. The cells exposed to unoxidized LDL, e.g. native LDL, acetyl‐LDL, and self‐aggregates of LDL showed no production of superoxide. The superoxide production was correlated with the levels of oxidative modification of LDL and reached a maximum between 10 and 30 min during incubation, but preincubating the cells with cytochalasin B decreased the superoxide production. These findings indicate that neutrophils rapidly take up ox‐LDL by phagocytosis and generate superoxide which may cause superoxide‐mediated lipid peroxidation in vivo.

Conformational changes in oxidized LDL recognized by mouse peritoneal macrophages.

Mouse peritoneal macrophages have been considered to recognize and take up oxidized LDL by a scavenger receptor. However, it is still unknown what conformational changes in oxidized LDL contribute to recognition by the macrophage scavenger receptor. In the present study, it was shown that the amount of oxidized LDL taken up by macrophages correlated well with the fluorescence intensity formed in oxidized LDL. The autofluorescent products generated in oxidized LDL were characterized by Ex:365 nm Em:430 nm, and the intensity of the fluorescence was reduced at base pH, and restored by adjusting the pH to neutral. The characteristics of the fluorescent products indicate that a Schiff base structure was formed in oxidized LDL. Oxidized LDLs were fractionated into native size and aggregated large particles with HPLC by monitoring fluorescence. It was demonstrated that macrophages ingest selectively or preferentially aggregated oxidized LDL, but not native size oxidized LDL. The incorporation of aggregated oxidized LDL was remarkably suppressed by heparin and cytochalasin B. These results suggest that mouse peritoneal macrophages recognize the conformational changes in oxidized LDL related to the formation of a Schiff base structure with increasing autofluorescence, and ingest selectively aggregated large particles in oxidized LDL in a phagocytic process.

Ethanolamine plasmalogen and cholesterol reduce the total membrane oxidizability measured by the oxygen uptake method.

To investigate the effects of ethanolamine plasmalogen, phosphatidylethanolamine, cholesterol, and alpha-tocopherol on the oxidizability of membranes, various large unilamellar vesicles (LUVs) including these lipids and antioxidant were examined for their total membrane oxidizabilities, evaluated as R(p)/R(i)(1/2) value (where R(p) is rate of oxygen consumption and R(i)(1/2) is the square root of rate of chain initiation) by the oxygen uptake method with water-soluble radical initiator and inhibitor. Incorporation of bovine brain ethanolamine plasmalogen (BBEP) into vesicles as well as cholesterol led to lower the total membrane oxidizability dose-dependently. The effect of BBEP was more efficient in the presence of cholesterol in vesicles. On the other hand, diacyl counterpart, egg yolk phosphatidylethanolamine, and a typical radical scavenger, alpha-tocopherol, had no effect on the membrane oxidizability. Alpha-tocopherol only prolonged an induction period dose-dependently in the present oxidizing system, suggesting a novel antioxidant mechanism of ethanolamine plasmalogens besides the action of scavenging radicals.

LIPID PEROXIDATION AND CEROID ACCUMULATION IN MACROPHAGES CULTURED WITH OXIDIZED LOW DENSITY LIPOPROTEIN

When mouse peritoneal macrophages were cultured with oxidized human low density lipoprotein (ox-LDL), storage of ceroid-like pigments was observed within the cells by light and fluorescent microscopy, and fluorescence spectrophotometry. The fluorescent products exhibit the characteristics of Schiff base structures, having a fluorescence maximum of 430 nm and an excitation maximum of 355 nm, which has been generally accepted with fluorescent lipid peroxidation products. Similar fluorescent products were isolated from the atherosclerotic lesions of the aged human artery. Ox-LDL was also intraperitoneally injected into guinea pigs to study an early stage of ceroid accumulation in macrophages. An early event in guinea pigs was the appearance of neutrophils. The findings from the model systems suggest that the ox-LDL in the artery wall is probable chemotactic for neutrophils as well as monocytes. We propose the hypothesis that the production of superoxide by neutrophils causes further lipid peroxidation of native LDL and then produces large amounts of oxidatively modified LDL which is the souse of ceroid pigment accumulated within the foam cells in human atherosclerotic lesions.

Serum choline plasmalogens, particularly those with oleic acid in sn-2, are associated with proatherogenic state

Serum plasmalogens (Pls) (1-O-alk-1’-enyl-2-acyl glycerophospholipids) are of particular interest for studies on metabolic disorders associated with oxidative stress and chronic inflammation. Serum levels of Pls are known to correlate positively with HDL-cholesterol (HDL-C); however, few studies have examined serum Pls molecular species in association with pathophysiological conditions and their clinical significance. To clarify these, we determined serum levels of individual ether glycerophospholipids in Japanese asymptomatic cohorts (n = 428; 362 male and 66 female subjects) by LC/MS/MS, and examined their correlations with clinical parameters. We found that the proportion of choline Pls (PlsCho) among total serum phospholipids was significantly lower in the male group over 40 years old and was associated with multiple risk parameters more strongly than HDL-C. The abundance of serum PlsCho with oleic acid (18:1) in sn-2 exhibited the strongest positive correlation with serum concentrations of adiponectin and HDL-C, while being inversely associated with waist circumference and the serum levels of TG and small dense LDL-cholesterol. The characterization of serum ether glycerophospholipids verified the specificity of PlsCho, particularly the ones with 18:1 in sn-2, as a sensitive biomarker for the atherogenic state.

Tysnd1 Deficiency in Mice Interferes with the Peroxisomal Localization of PTS2 Enzymes, Causing Lipid Metabolic Abnormalities and Male Infertility

Peroxisomes are subcellular organelles involved in lipid metabolic processes, including those of very-long-chain fatty acids and branched-chain fatty acids, among others. Peroxisome matrix proteins are synthesized in the cytoplasm. Targeting signals (PTS or peroxisomal targeting signal) at the C-terminus (PTS1) or N-terminus (PTS2) of peroxisomal matrix proteins mediate their import into the organelle. In the case of PTS2-containing proteins, the PTS2 signal is cleaved from the protein when transported into peroxisomes. The functional mechanism of PTS2 processing, however, is poorly understood. Previously we identified Tysnd1 (Trypsin domain containing 1) and biochemically characterized it as a peroxisomal cysteine endopeptidase that directly processes PTS2-containing prethiolase Acaa1 and PTS1-containing Acox1, Hsd17b4, and ScpX. The latter three enzymes are crucial components of the very-long-chain fatty acids β-oxidation pathway. To clarify the in vivo functions and physiological role of Tysnd1, we analyzed the phenotype of Tysnd1−/− mice. Male Tysnd1−/− mice are infertile, and the epididymal sperms lack the acrosomal cap. These phenotypic features are most likely the result of changes in the molecular species composition of choline and ethanolamine plasmalogens. Tysnd1−/− mice also developed liver dysfunctions when the phytanic acid precursor phytol was orally administered. Phyh and Agps are known PTS2-containing proteins, but were identified as novel Tysnd1 substrates. Loss of Tysnd1 interferes with the peroxisomal localization of Acaa1, Phyh, and Agps, which might cause the mild Zellweger syndrome spectrum-resembling phenotypes. Our data established that peroxisomal processing protease Tysnd1 is necessary to mediate the physiological functions of PTS2-containing substrates.

Ethanolamine plasmalogens protect cholesterol-rich liposomal membranes from oxidation caused by free radicals.

The aim of the present study is to investigate the effect of ethanolamine plasmalogens on the oxidative stability of cholesterol-rich membranes by comparing it with that of diacyl glycerophosphoethanolamine, using bovine brain ethanolamine plasmalogen (BBEP) or egg yolk phosphatidylethanolamine (EYPE)-containing large unilamellar vesicles (LUVs) and the water-soluble radical initiator AAPH. Electron microscopic observation and particle size measurement visually demonstrated that ethanolamine plasmalogens protect cholesterol-rich phospholipid bilayers from oxidative collapse. Lipid analyses suggested that the effect of ethanolamine plasmalogens in stabilizing membranes against oxidation is partly due to the antioxidative action of plasmalogens involved in scavenging radicals at vinyl ether linkage.

Improvement and validation of 125I-high-performance liquid chromatography method for determination of total human serum choline and ethanolamine plasmalogens

Background Serum plasmalogens (Pls) have gained interest in several clinical symptoms such as metabolic syndrome/atherosclerosis or Alzheimers disease possibly because of their antioxidant properties. We have developed a highly sensitive and simple method to determine plasmenylcholine (PlsCho; choline plasmalogen) and plasmenylethanolamine (PlsEtn; ethanolamine plasmalogen) separately, using a radioactive iodine and high-performance liquid chromatography (125I-HPLC method). The present study reports the improvement and validation of 125I-HPLC method by introducing a quantitative standard (QS) and online detection with a flow γ-counter. Methods 1-Alkenyl 2,3-cyclic glycerophosphate was prepared as QS from l-α-lyso plasmenylcholine by enzymatic treatment with phospholipase D. Online detection with a flow γ-counter was investigated to be available to quantify Pls. The method validation was carried out in terms of selectivity, sensitivity, linearity, precision, accuracy and recovery. Results Linearity was established over the concentration range 5–300 μmol/L for Pls and QS with regression coefficients >0.99. The accuracy and reliability were satisfactory. The method has been applied to the determination of human serum Pls from healthy subjects and the elderly with dementia or artery stenoses. Conclusions The improved 125I-HPLC method is useful as an autoanalytical system for a routine diagnostic test of human serum Pls.

Plasma/Serum Plasmalogens: Methods of Analysis and Clinical Significance.

Age-related diseases, such as atherosclerosis and dementia, are associated with oxidative stress and chronic inflammation. Peroxisome dysfunction may be related to aging and age-related pathologies, possibly through the derangement of redox homeostasis. The biosyntheses of plasmalogens (Pls), a subclass of glycerophospholipids, are primarily regulated by peroxisomes. Thus, plasma Pls may reflect the systemic functional activity of peroxisomes and serve as potential biomarkers for diseases related to oxidative stress and aging. Recently, we have established three promising analytical methods for plasma/serum Pls using high-performance liquid chromatography with radioactive iodine, liquid chromatography-tandem mass spectrometry, and enzymatic assay. These methods were validated and used to obtain detailed molecular information regarding these molecules. In cross-sectional studies on asymptomatic, coronary artery disease, and elderly dementia individuals, we found that serum choline Pls, particularly those containing oleic and linoleic acid in the sn-2 position of the glycerol backbone, may serve as reliable antiatherogenic biomarkers. Furthermore, we also found that serum ethanolamine Pls were effective in discriminating cognitive impairment. These results support our hypothesis and further studies are clearly needed to elucidate Pls pathophysiologic significance.

Accumulation of ceroid-like pigments in macrophages cultured with phosphatidylcholine liposomes in vitro

When mouse peritoneal macrophages as well as P388D1 cells, an established macrophage-like cell line, were cultured with liposomes composed of rat liver phosphatidylcholine and phosphatidylserine, storage of fluorescent products, ceroid-like pigments, within those cells was observed with light and fluorescence microscopy, and fluorescence spectrophotometry. The amounts of thiobarbituric acid-reactive substances and fluorescent products in macrophages were increased gradually to reach a maximal level to between 6 and 8 days of culture. The involvement of peroxidation of liposomal lipids in the formation of the pigments was further suggested by the 6 days that incorporation of alpha-tocopherol into liposomes decreased the storage of the pigments. No appreciable formation of the pigments was observed in macrophages cultured with liposomes containing dipalmitoylphosphatidylcholine instead of rat liver phosphatidylcholine. The fluorescent products formed in cultured cells were found in lipid-soluble and -insoluble fractions. Lipid-insoluble fluorescent products had an excitation maximum at 360 nm and a fluorescence maximum at 430 nm in SDS-aqueous solution (pH 7.4) and the intensity of the fluorescence was quenched at base pH, but it was not changed in acidic media. These findings indicate that the macrophages can store Schiff base fluorescent substances formed by the reaction between peroxidation products of exogenous lipids and amino compounds in the cells, under some pathological conditions.